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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0693—Tumour cells; Cancer cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2503/00—Use of cells in diagnostics
  • C12N2503/02—Drug screening
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the invention relates to methods for the preparation of a cell culture monolayer, and more particularly to methods for the preparation of a tumor cell culture monolayer that substantially comprises tumor cells.
• Kruczynski, A., et al. “Evidence of a direct relationship between the increase in the in vitro passage number of human non-small-cell lung cancer primocultures and their chemosensitivity,” Anticancer Research , vol. 13, no. 2, pp. 507-513 (1993)
• chemosensitivity of non-small-cell lung cancers was investigated in in vivo grafts, in in vitro primocultures, and in commercially available cancer cell lines. The increase in chemosensitivity was documented and correlated with morphological changes in the cells in question.
• the cells are harvested (biopsied) and trypsinized (connective tissue digested with the enzyme trypsin) to yield a cell suspension suitable for conversion to the desired tissue culture form.
• trypsinized connective tissue digested with the enzyme trypsin
• the in vitro tissue culture cell preparations typically fail to accurately replicate the chemosensitivity of the original tumor or other cell biopsy. This inability arises, in part, because the heterogeneity of cell population in the tumor tissue has been disturbed in culture, or entirely obliterated such that the cell culture preparation is essentially monoclonal.
• the present invention provides methods for preparing a cell culture from a multicellular tissue extract.
• Cell cultures of the invention have the advantage of closely resembling the in vivo cell population from which they were obtained, thus providing an accurate and reliable proxy for the cell population in vivo.
• a tumor cell culture of the invention comprises a population of cells that mimics the tumor cell population in the patient from whom a tissue explant is obtained. This allows chemosensitivity and chemoresistivity testing that is highly-reliable in predicting the effects of therapeutic agents on the tumor in vivo.
• the invention is based, in part, on the insight that timely removal of a cellular explant from culture results in a culture that is highly indicative of in vivo cell population.
• the invention provides further benefits recognized by culturing tissue explants in a growth medium that is essentially free of digestive enzymes.
• the invention provides a cell culture system in which a multicellular tissue explant is placed in a growth medium and is removed from the growth medium at a predetermined time.
• the explant is removed prior to the emergence from the explant of a substantial number of non-target cells, resulting in a monolayer of cells that is enriched for the cell population of interest.
• a monolayer of cells that is enriched for the cell population of interest.
• cells emerge as a monolayer from a cultured tumor tissue explant in an orderly fashion, the tumor cells emerging first, followed by stromal cell populations. If the tumor cell explant remains in culture, the stromal cells have been found to dominate the tumor cells in culture.
• the time at which an explant is removed from its culture medium depends upon the type of cells being cultured, the rate of emergence of various cell types, and the desired purity of the resulting cell culture monolayer. This can be determined empirically for a given cell type.
• the multicellular tissue explant is preferably removed when the cell culture monolayer is at about 10 to about 50 percent confluency. In a preferred embodiment, the multicellular tissue explant is removed at about 15 to about 25 percent confluency. In a particularly preferred embodiment, the explant is removed at about 20 percent confluency.
• the invention further comprises the preparation of a cell suspension from the cell culture monolayer.
• a tissue explant is cultured in an appropriate medium and is removed at a predetermined time, resulting in a monolayer enriched for the cells of interest.
• a suspension is then made from the monolayer and cells of the suspension are inoculated into at least one segregated site.
• a chemosensitivity assay is performed on the inoculated cell suspension by exposing the segregated site to at least one agent and assessing the chemosensitivity of the cells in the segregated site. Chemoresistivity assays are similarly performed.
• the invention provides methods for determining the chemosensitivity of a tissue in a patient by determining the chemosensitivity of a cell culture preparation from the patient. In yet another embodiment, the invention provides methods for identifying an agent having anti-tumorogenic effect in a patient by assessing the chemosensitivity of segregated sites of cells from a tumor cell culture prepared according to the invention.
• the present invention provides methods for preparing a cell culture monolayer by culturing a tissue sample from a patient.
• the culture may be used to screen at least one candidate therapeutic or chemotherapeutic agent for efficacy as to a specific patient, in which a tissue sample from the patient is harvested and separately exposed to a plurality of treatments and/or therapeutic agents for the purpose of objectively identifying the chemosensitivity or chemoresistivity of the tissue sample and the best treatment or agent for the patient.
• Tissue sample preparation techniques render this method practically as well as theoretically useful.
• the initial cohesive multicellular particulates (explants) of the tissue sample are prepared mechanically, rather than enzymatically, for initial tissue culture monolayer preparation.
• the multicellular tissue explant is removed from the culture growth medium at a predetermined time to both allow for the growth of target cells and prevent substantial growth of non-target cells such as fibroblasts or stromal cells.
• An important application of the present invention is the screening of chemotherapeutic agents and other antineoplastic or anti-tumorogenic therapies against tissue culture preparations of tumorogenic cells from the patient from whom the sample is biopsied.
• Related anti-cancer therapies which can be screened using the methods of the invention are both radiation therapy and agents which enhance the cytotoxicity of radiation, as well as immunotherapeutic anti-cancer agents. Screening processes for treatment or therapeutic agents for nonmalignant syndromes are also embraced within this invention, however, and include without limitation, agents which combat hyperproliferative diseases, such as psoriasis, or wound healing agents.
• present efficacy assay limited only to the screening of active agents which speed up (healing) or slow down (anti-cancer, anti-hyperproliferative) cell growth because agents intended to enhance or to subdue intracellular biochemical functions may be tested in the present tissue culture system as well.
• the formation or blocking of enzymes, neurotransmitters and other biochemicals may be screened with the present assay methods prior to treatment of the patient.
• a cell culture monolayer in accordance with the invention is prepared using the following procedure. Many aspects of the following procedure may be altered as necessary and as well known in the art.
• a biopsy of normecrotic, non-contaminated tissue is harvested from the patient by any suitable biopsy or surgical procedure known in the art.
• the tissue sample is tumor tissue. In one embodiment, the biopsy is at least about 100 mg.
• Biopsy sample preparation generally proceeds under sterile conditions.
• Cohesive multicellular particulates (explants) are prepared from the tissue sample using mechanical fragmentation. In one embodiment, this mechanical fragmentation of the explant occurs in a medium substantially free of enzymes that are capable of digesting the explant.
• the tissue sample is minced with sterile scissors to prepare the explants.
• the tissue sample is systematically minced by using two sterile scalpels in a scissor-like motion, or mechanically equivalent manual or automated opposing incisor blades. This cross-cutting motion creates smooth cut edges on the resulting tissue multicellular particulates. In one embodiment, multicellular particulates measuring about 1 mm 3 may be produced.
• the particles are plated in culture flasks (for example, 9 explants per T-25 or 20 particulates per T-75 flask). The explants may be evenly distributed across the bottom surface of the flask, followed by initial inversion for about 10-15 minutes.
• the flask may then be placed in a non-inverted position in a 37° C. CO 2 incubator for about 5-10 minutes.
• the tissue sample comprises brain cells
• the flasks are placed in a 35° C., non-CO 2 incubator. Flasks should be checked regularly for growth and contamination.
• the multicellular explant is removed from the cell culture at a predetermined time, as described below. Over a period of a few weeks a monolayer will be produced. With respect to the culturing of tumor cells, it is believed (without any intention of being bound by the theory) that tumor cells grow out from the multicellular explant prior to stromal cells.
• growth of the tumor cells (as opposed to stromal cells) into a monolayer is facilitated.
• tissue culture monolayer culture maximizes the growth of tumor cells from the tissue sample, and thus optimizes ensuing tissue culture assay of various agents (e.g., chemotherapeutic agents) to be tested.
• agents e.g., chemotherapeutic agents
• the growth of the cells may be monitored to oversee growth of the monolayer and ascertain the time to initiate the chemotherapy assay and to determine the growth rate of the cultured cells.
• monitoring of the growth of cells may be conducted by visual monitoring of the flasks on a periodic basis, without killing or staining the cells and without removing any cells from the culture flask.
• Data from periodic counting is then used to determine growth rates which may or may not be considered parallel to growth rates of the same cells in vivo in the patient. If growth rate cycles can be documented, for example, then dosing of certain active agents can be customized for the patient. The same growth rate can be used to evaluate radiation treatment periodicity, as well. It should be noted that with the growth rate determinations conducted while the monolayers grow in their flasks, the present method requires no hemocytometry, flow cytometry or use of microscope slides and staining, with all their concomitant labor and cost.
• Monolayer growth rate may be monitored using, for example, a phase-contrast inverted microscope.
• culture flasks are incubated in a (5% CO 2 ) incubator at about 37° C.
• the flask is placed under the phase-contrast inverted microscope, and ten fields (areas on a grid inherent to the flask) are examined using the 10 ⁇ objective.
• the ten fields should be non-contiguous, or significantly removed from one another, so that the ten fields are a representative sampling of the whole flask. Percentage cell occupancy for each field examined is noted, and averaging of these percentages then provides an estimate of overall percent confluency in the cell culture.
• an average cell count for the total patient sample should be calculated.
• the calculated average percent confluency should be entered into a process log to enable compilation of data—and plotting of growth curves—over time. Alternatively, confluency may be judged independently for each flask. Monolayer cultures may be photographed to document cell morphology and culture growth patterns.
• Percent confluency estimate of the area occupied by cells total area in an observed field
• the tissue explant is removed from the growth medium at a predetermined time.
• the explant is removed from the growth medium prior to the emergence of a substantial number of stromal cells from the explant.
• the explant may be removed according to the percent confluency of the cell culture.
• the explant is removed at about 10 to about 50 percent confluency.
• the explant is removed at about 15 to about 25 percent confluency.
• the explant is removed at about 20 percent confluency.
• a cell culture monolayer predominantly composed of target cells e.g., tumor cells
• target cells e.g., tumor cells
• non-target cells such as fibroblasts or other stromal cells
• this method of culturing a multicellular tissue explant and subsequently removing the explant at a predetermined time allows for increased efficiency in both the preparation of cell cultures and subsequent assays of various agents using the cultures.
• Adaptation of the above protocol for non-tumor cells is straightforward and generally constitutes an equivalent procedure.
• the essence of the invention thus includes the important feature of the simplicity of the present system—cohesive multicellular explants of the patient tissue to be tested are used to form cell monolayers; growth of those monolayers may be monitored for accurate prediction of correlating growth of the same cells in vivo; explants are removed from the growth medium at a predetermined time, and differing concentrations of a number of active agents may be tested for the purpose of determining chemosensitivity of the tissue sample and the most appropriate agent and concentration of that agent for actual patient exposure (according to the calculated cell growth rates).
• the present system allows in vitro tests to be conducted in suspensions of tissue culture monolayers grown in nutrient medium under fast conditions (a matter of weeks), rather than with single cell progeny produced by dilution cloning over long periods of time.
• the present invention provides a cell culture for a two stage assay for both cytotoxicity and the longer-term growth inhibitory.
• An initial step in preparing the microtiter plates is preparing and monitoring the monolayer as described above with the removal of the explant at 20 percent confluency.
• the following example shows an exemplary protocol which is susceptible of variation as will be apparent to one skilled in the art.
• Cells were removed from the culture flask and a cell pellet was prepared by centrifugation.
• the cell pellet derived from the monolayer was then suspended in 5 ml of the growth medium, mixed in a conical tube and subsequently rocked back and forth 10 times.
• a 30 ⁇ l droplet from the center of the conical tube was pipetted into one well of a 96 well plate.
• a fresh pipette was then used to pipette a 30 ⁇ l aliquot of trypan blue solution, which was added to the same well, and the two droplets were mixed with repeated pipette aspiration.
• the resulting admixture was then applied to a hemocytometer chamber for examination using a standard light microscope. Cells were counted in all of four hemocytometer quadrants, under 10 ⁇ magnification. Only those cells which had not taken up the trypan blue dye were counted. Using means known in the art, the quadrant count values were checked, logged, multiplied by 10 4 to give cells/ml, and the total amount of fluid (growth medium) necessary to suspend remaining cell aliquots was calculated accordingly.
• the following example provides an exemplary protocol for assaying active agents in accordance with the invention.
• the appropriate amount of specific active agent was transferred into the microtiter plates prepared as described above.
• the plates were blotted with sterile gauze to remove the agent, washed with Hank's Balance Salt Solution, flooded with growth medium, and replaced in the incubator in an incubator box for a predefined time period, after which the plates were fixed and stained for evaluation.
• Fixing and staining may be conducted according to a number of suitable procedures; the following is representative. After removal of the plates from the incubator box, culture medium were poured off and the plates were flooded with Hank's Balance Salt Solution. After repeated flooding (with agitation each time) the plates were then flooded with reagent grade ethanol for 2-5 minutes. The ethanol was then poured off. Staining was accomplished using a DAPI ( 4 ′, 6-diamidino-2-phenylindole, dilactate) staining method. Each plate was flooded with a DAPI/water solution, with a concentration of about 400 nM, and allowed to stand for at least 10 minutes, after which the DAPI/water was poured into a beaker.
• DAPI 4 ′, 6-diamidino-2-phenylindole, dilactate
• the plates were then dipped into a beaker of running water to remove the excess DAPI.
• Cells per well may then be counted manually or by automated and/or computerized means, to derive data regarding chemosensitivity of cells at various concentrations of exposure.
• One particularly useful computer operating environment for counting cells is the commercially available Zeiss Axiovert S100 Automatic Inverted Flourescence Microscope and Computer.
• the above procedures do not change appreciably when cell growth promoters are assayed rather than cell arresting agents such as chemotherapeutic agents.
• the present assay allows cell death or cell growth to be monitored with equal ease.
• optimization of use of the present system will involve the comparative testing of a variety of candidate active agents, for selection of the best candidate for patient treatment based upon the in vitro results.
• One particularly advantageous embodiment of the above-described invention comprises a two-stage assay for cytotoxicity followed by evaluation of longer-term inhibitory effect chemotherapeutic agents may thus be evaluated separately for both their direct chemotherapeutic effect as well as for their longer duration efficacy.

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• 2002
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